Elucidation of the mechanism of caediovascular remodeling and development of therapy by proteomic analysis

本研究的目的是阐明心血管重构的机制及其治疗进展。通过引入蛋白质组学研究，主要针对我们过去发现的在心血管重塑中起重要作用的转录因子KLF5，对影响心血管疾病发病过程的因子辅助因子进行了全面的分离，并从功能缺失系统的角度进行了功能分析。本研究最终以KLF5为模型，阐明了细胞核内转录因子网络的转录控制机制，旨在研究器官重塑的预防和治疗。PDGF-A促进平滑肌细胞的迁移/增加，是一种在动脉粥样硬化发生中起重要作用的生长因子。通过我们过去的研究发现它是KLF5的一个靶基因，并且清楚地表明-71到-55bp结构域在心血管重塑中很重要。首先，作为本研究的具体计划，我们鉴定结合PDGF-A基因-71 ~ -55bp结构域的蛋白。我对KLF5结合蛋白进行了筛选。具体来说，我们构建了-71至-55bp结构域的双链DNA，该结构域是PDGF-A启动子的KLF5结合位点，并将其一端生物素化。我们使用亲链素和生物素的强力结合，将其固定在金属珠表面。我把细胞的核萃取物放在这里，然后拉下来。然后，我们建立了SDS-PAGE，并通过凝胶内胰蛋白酶消化法鉴定了特定的条带。发现了一些有趣的因素。基于KLF5结合因子的生化特性，从动物水平到细胞水平探究其致病机制。由此，揭示了心血管组织抗氧化应激防御机制的新机制。

The objective of this study is to elucidate the mechanism of cardiovascular remodeling and the development of its therapy. By introducing research proteomics, and, mainly on transcription factor KLF5 which had an important role for the cardiovascular remodeling which we identified in the past, we perform a comprehensive isolation of the co-factors of the factor which affect the pathogenic process of the cardiovascular diseases, a functional analysis by the loss of function system. This study finally elucidates transcription control mechanism by the transcription factor network in the nucleus which assumed KLF5 a model and aims for developing the prevention of the organ remodeling and a cure. PDGF-A promotes a migration/the increase of the smooth muscle cell, and it is a growth factor playing an important role for the onset of the atherosclerosis. It was found to be a target gene of KLF5 by the examination of our past, and it became clear that -71 to -55bp domains were important in cardiovascular remodeling. At first, as a concrete plan in this study, we identify protein which bind -71 to -55bp domains of PDGF-A gene. I follow screening of the KLF5 binding protein basically. To be concrete, we composed the double-stranded DNA of -71 to -55bp domains that were the KLF5 binding site of the PDGF-A promoter and biotinylated the one end. Using streptoavidin and a strong bond of the biotin, we fixed it on the metal beads surface. I reacted the nuclear extract of the cell in this, and pull down. Then, we developed SDS-PAGE and identified a specific band by in-gel trypsin digestion method. Some interesting factors were identified. Based on the biochemical characteristic of the KLF5 binding factor, I pursued the pathogenic mechanism from in the animal level to the cell level. Above all, new mechanism in the defense mechanism of the cardiovascular tissues from oxidation stress became clear.